Rapid microbial detection is very important for the detection of foodborne pathogenic microorganisms, the detection of environmentally harmful bacteria, the detection of infectious bacteria, the diagnosis of pathogenic virus, and the like. Methods for determining the presence and concentration of pathogenic substances (microorganisms, proteins, etc.), which are generally frequently used, include a colony assay, a DNA probe assay, an immunoassay, and the like (Jay J M. Modern Food Microbiology, 1986, 3rd, ed., p 95, Van Nostrand Reinhold Co., New York; Tenover F C., DNA Probes for Infectious Diseases, 1989, p 53 CRC Press, Boca Raton).
In the colony assay, a sample is collected and incubated in a selective medium composed of components selected such that only the type of microorganisms to be detected can survive, after which the number of colonies by the microorganisms is measured. This assay method is very accurate, but has disadvantages in that a long time is required for measurement and it is difficult to select a medium for each type of microorganisms.
The DNA probe assay includes real-time PCR (polymerase chain reaction) and nucleic acid hybridization. In this method, microorganisms are physically and chemically destroyed, and then DNA in the microbial cells is detected by nucleic acid hybridization. Although this method has an advantage in that the test time is shorter than that in the colony assay method, it has disadvantages in that an expensive PCR system is used and when a small amount of microorganisms are to be detected, a separate culture step should be carried out in order to attain high sensitivity (Ninet, B et al., Appl Environ Microbiol, 58:4055-4059, 1992). If the culture step is not carried out, dead cells can be detected, resulting in incorrect results. In addition, when PCR is carried out, false-positive reactions can very frequently occur to increase the detection error and reduce the reliability of analysis.
The immunoassay method is based on an antigen-antibody binding reaction. For example, an enzyme-linked immunosorbent assay (ELISA) which uses an antibody specific to a surface antigen of the microorganisms to be detected is widely known. This method shows high sensitivity in a short time, and thus is considered as a substitute for the above-described two methods. In this method, an antibody can be continuously produced by a hybridoma, and thus the occurrence of problems can be minimized. However, such a immunoassay method has a drawback in that a highly pure antigen, an expensive system, and a long-term test are required.
Another antibody-based assay is an immunomagnetic separation (IMS) method, which can shorten enrichment time and can selectively capture bacteria by employing specific antibodies bound to magnetic particles or beads (Lynch, M J et al., J Microbiol Methods 58:285-288, 2004; Notermans, S et al., Int J Food Microbiol, 12:91-102, 1991). IMS is used to capture and concentrate selective target organisms, proteins, or nucleic acids (Favrin, S J et al., Int J Food Microbiol, 85:63-71, 2003; Feng P., ASM press, Washington D.C., 2001). Like other antibody-based assays, however, IMS also requires an enrichment process and is limited for use on small volume samples.
SPR sensor technology is a method of detecting microorganisms using a phenomenon in which a signal change occurs when biomaterials such as proteins are bound to the sensor surface. This method requires a short detection time and a simple assay procedure, compared to other assay methods. However, because the size of pathogenic microorganisms in a sample is much larger than an antibody immobilized on the sensor surface, the efficiency of binding of the microorganisms to the antibody by an immune response in a fluid flow in the SPR system is low. As a result, due to the inefficient immune reaction of the antibody and the immobilization of the antibody on the sensor surface, the detection limit of pathogenic microorganisms is disadvantageously high.
Accordingly, the present inventors have made extensive efforts to solve the above-described problems and, as a result, have found that the high-sensitivity detection and analysis of microorganisms can be achieved using not only an immune reaction with nanoparticles, which have immobilized thereon an antibody that binds specifically to the microorganisms to be detected, but also a membrane filtration method, thereby completing the present invention.